Abstract

We describe a dual, second harmonic generation (SHG) and third harmonic generation (THG) microscope, with the aim to obtain large-scale images of the cornea that can simultaneously resolve the micron-thick thin layers. We use an Ytterbium femtosecond laser as the laser source, the longer wavelength of which reduces scattering and allows simultaneous SHG and THG imaging. We measure one-dimensional SHG and THG profiles across the entire thickness of pig cornea, detected in both the forward and backward directions. These profiles allow us to clearly distinguish all the porcine corneal layers (epithelium, stroma, Descemet’s membrane and endothelium). From these profiles, longitudinal cross sectional images of the corneal layers are generated, providing large scale topographic information with high-spatial resolution. The ability to obtain both SHG and THG signals in epi-detection on fresh eyes gives promising hopes for in vivo applications.

© 2008 Optical Society of America

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  1. A. C. M. Wong, C. C. Wong, N. S. Y. Yuen, and S. P. Hui, "Correlational study of central corneal thickness measurements on Hong Kong Chinese using optical coherence tomography, Orbscan and ultrasound pachymetry," Eye 16, 715 (2002).
    [CrossRef] [PubMed]
  2. A. P. Adamis, V. Filatov, B. J. Tripathi, and R. C. Tripathi, "Fuch??s endothelial dystrophy of the cornea," Surv. Ophthalmol. 38, 149-163 (1993).
    [CrossRef] [PubMed]
  3. I. Jalbert, F. Stapleton, E. Papas, D. F. Sweeney, and M. Coroneo, "In vivo confocal microscopy of the human cornea," Br. J. Ophthalmol. 87, 225-236 (2003).
    [CrossRef] [PubMed]
  4. S. Fine and W. P. Hansen, "Optical second harmonic generation in biological systems," Appl. Opt. 10, 2350 (1971).
    [CrossRef] [PubMed]
  5. N. Morishige, W. M. Petroll, T. Nishida, M. C. Kenney, and J. V. Jester, "Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals," J. Cataract Refract. Surg. 32, 1784 (2006).
    [CrossRef] [PubMed]
  6. Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922 (1997).
    [CrossRef]
  7. D. Yelin and Y. Silberberg, "Laser scanning third-harmonic-generation microscopy in biology," Opt. Express 5, 169-175 (1999).
    [CrossRef] [PubMed]
  8. W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
    [CrossRef] [PubMed]
  9. B. G. Wang, A. Eitner, J. Lindenau, and K. J. Halbhuber, "High-Resolution Two-Photon Excitation Microscopy of Ocular Tissues in Porcine Eye," Lasers Surg. Med. 40, 247-256 (2008).
    [CrossRef] [PubMed]
  10. S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
    [CrossRef] [PubMed]
  11. E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, "In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy," J. Microsc. 229, 141-150 (2008).
    [CrossRef] [PubMed]
  12. E. Svaldeniene, V. Babrauskiene, and M. Paunksniene, "Structural features of the cornea: light and electron microscopy," Veterinarija ir Zootechnika 46, 50-55 (2003).
  13. A. Brocas, L. Jay, E. Mottay, I. Brunette, and T. Ozaki, "Corneal imaging by second and third harmonic generation microscopy," Proc. SPIE 6860, 68600C (2008).
    [CrossRef]
  14. J. Squier and M. Müller, "High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging," Rev. Sci. Instrum. 72, 2855-2867 (2001).
    [CrossRef]
  15. K. M. Meek, S. Dennis, and S. Khan, "Changes in the Refractive Index of the Stroma and Its Extrafibrillar Matrix When the Cornea Swells," Biophys. J. 85, 2205-2212 (2003).
    [CrossRef] [PubMed]

2008

B. G. Wang, A. Eitner, J. Lindenau, and K. J. Halbhuber, "High-Resolution Two-Photon Excitation Microscopy of Ocular Tissues in Porcine Eye," Lasers Surg. Med. 40, 247-256 (2008).
[CrossRef] [PubMed]

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, "In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy," J. Microsc. 229, 141-150 (2008).
[CrossRef] [PubMed]

A. Brocas, L. Jay, E. Mottay, I. Brunette, and T. Ozaki, "Corneal imaging by second and third harmonic generation microscopy," Proc. SPIE 6860, 68600C (2008).
[CrossRef]

2006

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

N. Morishige, W. M. Petroll, T. Nishida, M. C. Kenney, and J. V. Jester, "Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals," J. Cataract Refract. Surg. 32, 1784 (2006).
[CrossRef] [PubMed]

2003

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

I. Jalbert, F. Stapleton, E. Papas, D. F. Sweeney, and M. Coroneo, "In vivo confocal microscopy of the human cornea," Br. J. Ophthalmol. 87, 225-236 (2003).
[CrossRef] [PubMed]

E. Svaldeniene, V. Babrauskiene, and M. Paunksniene, "Structural features of the cornea: light and electron microscopy," Veterinarija ir Zootechnika 46, 50-55 (2003).

K. M. Meek, S. Dennis, and S. Khan, "Changes in the Refractive Index of the Stroma and Its Extrafibrillar Matrix When the Cornea Swells," Biophys. J. 85, 2205-2212 (2003).
[CrossRef] [PubMed]

2002

A. C. M. Wong, C. C. Wong, N. S. Y. Yuen, and S. P. Hui, "Correlational study of central corneal thickness measurements on Hong Kong Chinese using optical coherence tomography, Orbscan and ultrasound pachymetry," Eye 16, 715 (2002).
[CrossRef] [PubMed]

2001

J. Squier and M. Müller, "High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging," Rev. Sci. Instrum. 72, 2855-2867 (2001).
[CrossRef]

1999

1997

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

1993

A. P. Adamis, V. Filatov, B. J. Tripathi, and R. C. Tripathi, "Fuch??s endothelial dystrophy of the cornea," Surv. Ophthalmol. 38, 149-163 (1993).
[CrossRef] [PubMed]

1971

Adamis, A. P.

A. P. Adamis, V. Filatov, B. J. Tripathi, and R. C. Tripathi, "Fuch??s endothelial dystrophy of the cornea," Surv. Ophthalmol. 38, 149-163 (1993).
[CrossRef] [PubMed]

Babrauskiene, V.

E. Svaldeniene, V. Babrauskiene, and M. Paunksniene, "Structural features of the cornea: light and electron microscopy," Veterinarija ir Zootechnika 46, 50-55 (2003).

Barad, Y.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Brocas, A.

A. Brocas, L. Jay, E. Mottay, I. Brunette, and T. Ozaki, "Corneal imaging by second and third harmonic generation microscopy," Proc. SPIE 6860, 68600C (2008).
[CrossRef]

Brunette, I.

A. Brocas, L. Jay, E. Mottay, I. Brunette, and T. Ozaki, "Corneal imaging by second and third harmonic generation microscopy," Proc. SPIE 6860, 68600C (2008).
[CrossRef]

Coroneo, M.

I. Jalbert, F. Stapleton, E. Papas, D. F. Sweeney, and M. Coroneo, "In vivo confocal microscopy of the human cornea," Br. J. Ophthalmol. 87, 225-236 (2003).
[CrossRef] [PubMed]

Dennis, S.

K. M. Meek, S. Dennis, and S. Khan, "Changes in the Refractive Index of the Stroma and Its Extrafibrillar Matrix When the Cornea Swells," Biophys. J. 85, 2205-2212 (2003).
[CrossRef] [PubMed]

Dong, C. Y.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

Eisenberg, H.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Eitner, A.

B. G. Wang, A. Eitner, J. Lindenau, and K. J. Halbhuber, "High-Resolution Two-Photon Excitation Microscopy of Ocular Tissues in Porcine Eye," Lasers Surg. Med. 40, 247-256 (2008).
[CrossRef] [PubMed]

Filatov, V.

A. P. Adamis, V. Filatov, B. J. Tripathi, and R. C. Tripathi, "Fuch??s endothelial dystrophy of the cornea," Surv. Ophthalmol. 38, 149-163 (1993).
[CrossRef] [PubMed]

Filippidis, G.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, "In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy," J. Microsc. 229, 141-150 (2008).
[CrossRef] [PubMed]

Fine, S.

Fotakis, C.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, "In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy," J. Microsc. 229, 141-150 (2008).
[CrossRef] [PubMed]

Gualda, E. J.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, "In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy," J. Microsc. 229, 141-150 (2008).
[CrossRef] [PubMed]

Halbhuber, K. J.

B. G. Wang, A. Eitner, J. Lindenau, and K. J. Halbhuber, "High-Resolution Two-Photon Excitation Microscopy of Ocular Tissues in Porcine Eye," Lasers Surg. Med. 40, 247-256 (2008).
[CrossRef] [PubMed]

Hansen, W. P.

Horowitz, M.

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

Hui, S. P.

A. C. M. Wong, C. C. Wong, N. S. Y. Yuen, and S. P. Hui, "Correlational study of central corneal thickness measurements on Hong Kong Chinese using optical coherence tomography, Orbscan and ultrasound pachymetry," Eye 16, 715 (2002).
[CrossRef] [PubMed]

Jalbert, I.

I. Jalbert, F. Stapleton, E. Papas, D. F. Sweeney, and M. Coroneo, "In vivo confocal microscopy of the human cornea," Br. J. Ophthalmol. 87, 225-236 (2003).
[CrossRef] [PubMed]

Jay, L.

A. Brocas, L. Jay, E. Mottay, I. Brunette, and T. Ozaki, "Corneal imaging by second and third harmonic generation microscopy," Proc. SPIE 6860, 68600C (2008).
[CrossRef]

Jee, S. H.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

Jester, J. V.

N. Morishige, W. M. Petroll, T. Nishida, M. C. Kenney, and J. V. Jester, "Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals," J. Cataract Refract. Surg. 32, 1784 (2006).
[CrossRef] [PubMed]

Kenney, M. C.

N. Morishige, W. M. Petroll, T. Nishida, M. C. Kenney, and J. V. Jester, "Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals," J. Cataract Refract. Surg. 32, 1784 (2006).
[CrossRef] [PubMed]

Khan, S.

K. M. Meek, S. Dennis, and S. Khan, "Changes in the Refractive Index of the Stroma and Its Extrafibrillar Matrix When the Cornea Swells," Biophys. J. 85, 2205-2212 (2003).
[CrossRef] [PubMed]

Kim, K. H.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

Lin, H. H.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

Lin, S. J.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

Lin, W. C.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

Lindenau, J.

B. G. Wang, A. Eitner, J. Lindenau, and K. J. Halbhuber, "High-Resolution Two-Photon Excitation Microscopy of Ocular Tissues in Porcine Eye," Lasers Surg. Med. 40, 247-256 (2008).
[CrossRef] [PubMed]

Lo, W.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

Mari, M.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, "In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy," J. Microsc. 229, 141-150 (2008).
[CrossRef] [PubMed]

Meek, K. M.

K. M. Meek, S. Dennis, and S. Khan, "Changes in the Refractive Index of the Stroma and Its Extrafibrillar Matrix When the Cornea Swells," Biophys. J. 85, 2205-2212 (2003).
[CrossRef] [PubMed]

Morishige, N.

N. Morishige, W. M. Petroll, T. Nishida, M. C. Kenney, and J. V. Jester, "Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals," J. Cataract Refract. Surg. 32, 1784 (2006).
[CrossRef] [PubMed]

Mottay, E.

A. Brocas, L. Jay, E. Mottay, I. Brunette, and T. Ozaki, "Corneal imaging by second and third harmonic generation microscopy," Proc. SPIE 6860, 68600C (2008).
[CrossRef]

Müller, M.

J. Squier and M. Müller, "High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging," Rev. Sci. Instrum. 72, 2855-2867 (2001).
[CrossRef]

Nishida, T.

N. Morishige, W. M. Petroll, T. Nishida, M. C. Kenney, and J. V. Jester, "Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals," J. Cataract Refract. Surg. 32, 1784 (2006).
[CrossRef] [PubMed]

Ozaki, T.

A. Brocas, L. Jay, E. Mottay, I. Brunette, and T. Ozaki, "Corneal imaging by second and third harmonic generation microscopy," Proc. SPIE 6860, 68600C (2008).
[CrossRef]

Papas, E.

I. Jalbert, F. Stapleton, E. Papas, D. F. Sweeney, and M. Coroneo, "In vivo confocal microscopy of the human cornea," Br. J. Ophthalmol. 87, 225-236 (2003).
[CrossRef] [PubMed]

Paunksniene, M.

E. Svaldeniene, V. Babrauskiene, and M. Paunksniene, "Structural features of the cornea: light and electron microscopy," Veterinarija ir Zootechnika 46, 50-55 (2003).

Peng, J. L.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

Petroll, W. M.

N. Morishige, W. M. Petroll, T. Nishida, M. C. Kenney, and J. V. Jester, "Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals," J. Cataract Refract. Surg. 32, 1784 (2006).
[CrossRef] [PubMed]

Silberberg, Y.

D. Yelin and Y. Silberberg, "Laser scanning third-harmonic-generation microscopy in biology," Opt. Express 5, 169-175 (1999).
[CrossRef] [PubMed]

Y. Barad, H. Eisenberg, M. Horowitz, and Y. Silberberg, "Nonlinear scanning laser microscopy by third harmonic generation," Appl. Phys. Lett. 70, 922 (1997).
[CrossRef]

So, P. T. C.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

Squier, J.

J. Squier and M. Müller, "High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging," Rev. Sci. Instrum. 72, 2855-2867 (2001).
[CrossRef]

Stapleton, F.

I. Jalbert, F. Stapleton, E. Papas, D. F. Sweeney, and M. Coroneo, "In vivo confocal microscopy of the human cornea," Br. J. Ophthalmol. 87, 225-236 (2003).
[CrossRef] [PubMed]

Sun, Y.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

Svaldeniene, E.

E. Svaldeniene, V. Babrauskiene, and M. Paunksniene, "Structural features of the cornea: light and electron microscopy," Veterinarija ir Zootechnika 46, 50-55 (2003).

Sweeney, D. F.

I. Jalbert, F. Stapleton, E. Papas, D. F. Sweeney, and M. Coroneo, "In vivo confocal microscopy of the human cornea," Br. J. Ophthalmol. 87, 225-236 (2003).
[CrossRef] [PubMed]

Tan, H. Y.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

Tavernarakis, N.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, "In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy," J. Microsc. 229, 141-150 (2008).
[CrossRef] [PubMed]

Teng, S. W.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
[CrossRef] [PubMed]

Tripathi, B. J.

A. P. Adamis, V. Filatov, B. J. Tripathi, and R. C. Tripathi, "Fuch??s endothelial dystrophy of the cornea," Surv. Ophthalmol. 38, 149-163 (1993).
[CrossRef] [PubMed]

Tripathi, R. C.

A. P. Adamis, V. Filatov, B. J. Tripathi, and R. C. Tripathi, "Fuch??s endothelial dystrophy of the cornea," Surv. Ophthalmol. 38, 149-163 (1993).
[CrossRef] [PubMed]

Voglis, G.

E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, "In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy," J. Microsc. 229, 141-150 (2008).
[CrossRef] [PubMed]

Wang, B. G.

B. G. Wang, A. Eitner, J. Lindenau, and K. J. Halbhuber, "High-Resolution Two-Photon Excitation Microscopy of Ocular Tissues in Porcine Eye," Lasers Surg. Med. 40, 247-256 (2008).
[CrossRef] [PubMed]

Webb, W. W.

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

Williams, R. M.

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
[CrossRef] [PubMed]

Wong, A. C. M.

A. C. M. Wong, C. C. Wong, N. S. Y. Yuen, and S. P. Hui, "Correlational study of central corneal thickness measurements on Hong Kong Chinese using optical coherence tomography, Orbscan and ultrasound pachymetry," Eye 16, 715 (2002).
[CrossRef] [PubMed]

Wong, C. C.

A. C. M. Wong, C. C. Wong, N. S. Y. Yuen, and S. P. Hui, "Correlational study of central corneal thickness measurements on Hong Kong Chinese using optical coherence tomography, Orbscan and ultrasound pachymetry," Eye 16, 715 (2002).
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A. C. M. Wong, C. C. Wong, N. S. Y. Yuen, and S. P. Hui, "Correlational study of central corneal thickness measurements on Hong Kong Chinese using optical coherence tomography, Orbscan and ultrasound pachymetry," Eye 16, 715 (2002).
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W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
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K. M. Meek, S. Dennis, and S. Khan, "Changes in the Refractive Index of the Stroma and Its Extrafibrillar Matrix When the Cornea Swells," Biophys. J. 85, 2205-2212 (2003).
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I. Jalbert, F. Stapleton, E. Papas, D. F. Sweeney, and M. Coroneo, "In vivo confocal microscopy of the human cornea," Br. J. Ophthalmol. 87, 225-236 (2003).
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Eye

A. C. M. Wong, C. C. Wong, N. S. Y. Yuen, and S. P. Hui, "Correlational study of central corneal thickness measurements on Hong Kong Chinese using optical coherence tomography, Orbscan and ultrasound pachymetry," Eye 16, 715 (2002).
[CrossRef] [PubMed]

Invest. Ophthalmol. Vis. Sci.

S. W. Teng, H. Y. Tan, J. L. Peng, H. H. Lin, K. H. Kim, W. Lo, Y. Sun, W. C. Lin, S. J. Lin, S. H. Jee, P. T. C. So, and C. Y. Dong, "Multiphoton autofluorescence and second-harmonic generation imaging of the ex vivo porcine eye," Invest. Ophthalmol. Vis. Sci. 47, 1216-1224 (2006).
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J. Cataract Refract. Surg.

N. Morishige, W. M. Petroll, T. Nishida, M. C. Kenney, and J. V. Jester, "Noninvasive corneal stromal collagen imaging using two-photon-generated second-harmonic signals," J. Cataract Refract. Surg. 32, 1784 (2006).
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E. J. Gualda, G. Filippidis, G. Voglis, M. Mari, C. Fotakis, and N. Tavernarakis, "In vivo imaging of cellular structures in Caenorhabditis elegans by combined TPEF, SHG and THG microscopy," J. Microsc. 229, 141-150 (2008).
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B. G. Wang, A. Eitner, J. Lindenau, and K. J. Halbhuber, "High-Resolution Two-Photon Excitation Microscopy of Ocular Tissues in Porcine Eye," Lasers Surg. Med. 40, 247-256 (2008).
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Nat. Biotechnol.

W. R. Zipfel, R. M. Williams, and W. W. Webb, "Nonlinear magic: multiphoton microscopy in the biosciences," Nat. Biotechnol. 21, 1369-1377 (2003).
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Opt. Express

Proc. SPIE

A. Brocas, L. Jay, E. Mottay, I. Brunette, and T. Ozaki, "Corneal imaging by second and third harmonic generation microscopy," Proc. SPIE 6860, 68600C (2008).
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A. P. Adamis, V. Filatov, B. J. Tripathi, and R. C. Tripathi, "Fuch??s endothelial dystrophy of the cornea," Surv. Ophthalmol. 38, 149-163 (1993).
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E. Svaldeniene, V. Babrauskiene, and M. Paunksniene, "Structural features of the cornea: light and electron microscopy," Veterinarija ir Zootechnika 46, 50-55 (2003).

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Figures (7)

Fig. 1.
Fig. 1.

(a) Experimental setup showing the laser source (t-pulse) and the home-built microscope for SHGI and THGI. In this illustration SHG is detected in transmission (forward) and THG in epi- (backward) detection. The PMTs are the photomultiplier tubes detecting both signals. (b) The LUMplanFL water immersion objective (Olympus) is used to focalize on cornea through the ophthalmic gel. The sample is a whole anterior segment (cornea,; aqueous humour,; iris,; lens) of a pig eye on a microscope slide, which sufficiently maintains the natural curved shape of the cornea.

Fig. 2.
Fig. 2.

1D B-SHG/F-THG profile of a pig cornea in the z-direction observed with a 63×/NA 0.75 Zeiss Plan Neofluar dry objective (Carl Zeiss MicroImaging) and measured up to a depth of 300 µm. No THG signal is detected after the air/epithelium interface. z-increment: 0.5 µm

Fig. 3.
Fig. 3.

(a) 1D profile (B-SHG/F-THG) at position x=60 µm in Fig. 6 along the full thickness of a pig cornea, observed with the 60×/NA 0.9 LUMplanFL water immersion objective (Olympus) and ophthalmic gel. Six sources of emission can be identified for the THG signal: (1) the gel-epithelium interface; (2) the epithelium-stroma interface; (3) the multiple interfaces within the stroma; (4) the stroma-Descemet’s membrane interface; (5) the Descemet’s membrane-endothelium interface; and (6) the endothelium-aqueous interface. Ultrasound pachymetry gives 922±3µm (b) Zoom view of the region encircled in red in Fig. 3(a) showing the posterior layers. z-increment: 1µm.

Fig. 4.
Fig. 4.

(a) 1D profile (F-SHG/B-THG) along the thickness of pig cornea with the 60×/NA 0.9 LUMplanFL water immersion objective (Olympus) and ophthalmic gel. The same six sources of emission as in Fig. 3 can be identified for the THG signal (from 1 to 6 in red). Ultrasound pachymetry gives 1013 ± 10 µm (b) Zoom view of the region encircled in red in Fig. 4(a) showing the posterior layers. z-increment: 1 µm

Fig. 5.
Fig. 5.

(a) 2D topographic profile combining B-SHG/F-THG (green/purple) along the whole thickness of the pig cornea. Scan increments of 60 µm in x-direction and 1µm in z-direction. Ultrasound pachymetry: 922±3µm. (b) Histology section of a pig cornea. Paraffin section stained with Masson’s trichrome.

Fig. 6.
Fig. 6.

(a) Histology section of the anterior portion of a pig cornea (Paraffin section stained with Masson’s trichrome). The superficial, wing, and basal epithelial cell layers can be distinguished. (b) Zoom from Fig. 5 (a) showing the non-parallel interfaces: gel/cornea and epithelium/stroma. (c) B-THG/F-SHG (purple/green) imaging of epithelial cells showing, as in the histological section, the different cellular sub-types. Scan increments were 400 nm in both direction (x,z).

Fig. 7.
Fig. 7.

(a) Histology section of the posterior part of a pig cornea showing the keratocytes (k) in the stroma, Descemet’s membrane (dm) and the endothelial layer (ec). (Paraffin section stained with Masson’s trichrome). (b) B-SHG/F-THG (green/purple) and (c) F-THG only imaging of the same layers. 400 nm-scan increments in both directions (x, z).

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